Despite widespread screening and routine intrapartum antimicrobial prophylaxis, group B Streptococcus (GBS) remains a major cause of infectious morbidity and mortality among infants. Screening programs involve substantial effort, cost, and antibiotic use and are not fully effective due to missed opportunities for prevention. For those reasons, the development of a GBS vaccine to prevent neonatal disease is a high priority, and candidate capsular polysaccharide conjugate vaccines are currently in human trials. These candidate vaccines target a subset of the known GBS serotypes, chosen based on epidemiologic studies of rectovaginal carriage (which is the reservoir for GBS transmission) and invasive disease. However, serotype distribution data are based largely on biochemical or immunologic assays of single colonies from clinical samples. We have developed a new real-time PCR-based serotyping assay that can detect multiple serotypes in complex biological samples, even when those serotypes are present at highly divergent concentrations. Based on our preliminary data, we hypothesize that carriage of non-vaccine GBS serotypes is substantially more common than previously realized, setting the stage for serotype replacement following conjugate vaccine introduction. Here we propose three specific aims designed to test hypotheses regarding the prevalence of colonization and co-colonization of GBS serotypes in human samples (Aim 1), to evaluate potential mechanisms by which GBS serotype replacement might occur using murine colonization models (Aim 2), and to use newly developed techniques for GBS genome manipulation to understand the role of specific capsular types in colonization density and co- colonization dynamics (Aim 3). The results of these studies will have relevance to GBS epidemiology, to mechanisms of carriage, transmission, and disease, and to understanding serotype replacement, which is a threat to conjugate vaccine programs beyond GBS.